The long term objective of the research projects in this proposal is to understand the molecular basis of signal transduction pathways in higher plants. Intra- and intercellular signal transductions are key processes that regulate cell growth, division and differentiation. Recent studies using natural products as probes have revealed many new signaling events in mammalian cells. One family of such natural products are immunosuppressive drugs cyclosporin A (CsA), FK5O6 and rapamycin. These drugs bind to their cellular receptors (referred to as immunophilins) and form receptor-ligand complexes that in turn target signaling molecules required for T cell activation therefore suppressing the immune response. In the absence of the drugs, immunophilins have been implicated in various cellular processes including protein folding and trafficking in both animal and yeast systems. In higher plant cells, as in T cells, certain signaling pathways are interrupted by immunosuppressive drugs. These pathways, unlike those in T cells, are initiated by plant hormones and modulate ion channel activities that control stomatal opening. In addition, plants produce a unique set of immunophilins that are localized in various subcellular compartments. In particular, two of the immunophilins are specifically expressed in green tissues and are localized in the chloroplast. These findings have uncovered a new family of proteins from higher plants and opened new possibilities of using immunosuppressive drugs as powerful probes to study intracellular signaling in plant cells. The specific objective of the proposed research is to elucidate the molecular properties and cellular functions of plant immunophilins and to identify the signaling components from plants that are targeted by immunophilin-ligand complexes. Towards these goals, cDNAs for plant immunophilins will be isolated and characterized. Detailed analyses of spatial and temporal patterns of immunophilin expression will be performed to provide clues on the functional significance of these proteins during plant development. Three approaches will be exploited to address the function of immunophilins in plant cell and development. One is to search for the partner proteins for immunophilins using affinity chromatography and yeast two-hybrid system. The second approach is to generate transgenic plants that are altered in the expression level of immunophilins and to monitor the phenotypic, cellular, and molecular changes of the plants (so called reverse genetic analysis). The last approach will utilize cell free systems to test whether plant immunophilins are involved in protein folding and trafficking as some of animal and yeast homologues. As a key step to dissect the immunosuppressants-sensitive signaling pathways in plant cells, the functional targets for these drugs will be identified and characterized at the molecular level . These studies will not only reveal the multifaceted nature of plant immunophilins but should also provide insight into immunophilin functions in mammalian cells.